Oncogenic herpesvirus hijacks components of the DNA repair ...
Transcript of Oncogenic herpesvirus hijacks components of the DNA repair ...
Oncogenic herpesvirus hijacks components of the DNArepair machinery to promote its own replication
Xiaofan Li, Ph.D.Bhaduri-McIntosh laboratory
Phosphorylation of Krüppel -associated Box (KRAB)-associated protein (KAP)-1by ATM is a key event during DDR
Yael Ziv et al., Nature Cell Biology, 2006
Krüppel -associated Box (KRAB)-associated protein (KAP)-1
376 493 628 801 835
651RBCC
BromoHP1 box
KAP1/TRIM28 (tripartite motif-containing protein 28) structure
KAP1 recruits HDACs and HMT to modify the epigenetic status around its binding locus
KAP1
DAPI
PS824
NuRD
CHD HDAC1/2
SETDB1
PS824
H3-K9 trimethylation H3-acetylation
KAP1 facilitates repair of DNA breaks in heterochromatin by allowing access to repair factors
Epstein-Barr virus (EBV)
Member of human gammaherpesvirus family
Enveloped, encapsidated large dsDNA genome
Mainly lymphoid, epithelial tropism
Associated with numerous human malignanciesBurkitt lymphoma Nasopharyngeal cell carcinoma Hodgkin’s diseasePost-transplant Lymphoproliferative diseases (PTLD)NK/T-Cell lymphomasGastric carcinoma
A. B.
C. D.
In situ hybridization for Epstein-Barr virus–encoded small RNAs (EBERs) inA. Burkitt lymphoma, B. Nasopharyngeal cell carcinoma, C. Hodgkin’s disease; D. PTLD
Lytic (re)activationLatent infection
Latency to lytic switch of EBV
Limited viral genes expressed
Viral genome duplicates in S-phase
No progeny production
HDACi, TGF-β, hypoxia, etc
ImmediateEarly
Early Late
Cellular components that regulate latency-to-lytic switch
-STAT3
-PCBP2
-KRAB-ZFPs: large family of transcriptional repressors
thought to function via the KAP1 corepressor
Derek Daigle, et al., JVI, 2010; Erik Hill, et al., JVI, 2013;
Siva Koganti, et al., JVI, 2015;
Mechanisms underlying EBV latency to lytic switch
Manipulation of KAP1 levels modulates EBV lytic activation
CA
E
0.5
1.0
1.5
0
0.5
1.0
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1.0
1.5
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BZ
LF
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RN
A
(rela
tive
am
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BM
RF
1m
RN
A
(rela
tive
am
ou
nt)
BF
RF
3m
RN
A
(rela
tive
am
ou
nt)
ZEBRA
KAP1
β-actin
0h 24h 48h 72h
Empty vector FLAG-KAP1
0h 24h 48h 72h
50kD
98kD
36kD1.00 0.85 0.66 0.36
D F
0h 13h 26h 39h
scrambled
ZEBRA
KAP1
β-actin
0h 13h 26h 39h
si-KAP1
50kD
98kD
36kD1.00 3.771.90 1.35
0.5
1.0
1.5
2.0
0
BZ
LF
1m
RN
A
(rela
tive
am
ou
nt)
BM
RF
1m
RN
A
(rela
tive
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BF
RF
3m
RN
A
(rela
tive
am
ou
nt)
0
1.5
*
3.0
4.5
6.0
0
1
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1.0
1.5
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EB
V D
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(rela
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B
**
*
**
*
*
scrambled
si-KAP1
VectorFLAG-KAP1
Xiaofan Li, et al., PLOS Pathogens, 2017
A
p-S824 KAP1
KAP1
β-actin
ZEBRA
EA-D
98kD
98kD
36kD
50kD
50kD
NaB (mM) 0 0.375 0.75 1.5 3
p-S824 KAP1
KAP1
β-actin
ZEBRA
EA-D
98kD
98kD
36kD
50kD
50kD
Dox (μg/ml) 0 0.15 0.6 2.5 10
p-S824 KAP1 ZEBRA DAPI
p-S824 KAP1 EA-D DAPI
B
D E
F
100
101
102
103
104
100
101
102
103
104
006
FL1-H
FL
4-H
0.778 40.2
6.8252.2
100
101
102
103
104
100
101
102
103
104
005
FL1-H
FL
4-H
1.04 0.846
0.26597.9
100
101
102
103
104
100
101
102
103
104
010
FL1-H
FL
4-H
1.04 39.6
5.0454.3
100
101
102
103
104
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101
102
103
104
009
FL1-H
FL
4-H
0.907 1.07
1.296.8
1.04 0.846
97.9 0.265
0.778 40.2
52.2 6.82
0.907 1.07
96.8 1.2
1.04 39.6
54.3 5.04
ZEBRA
p-S
824 K
AP
1
EAD
p-S
824 K
AP
1
Dox- Dox+
EAD
100
101
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104
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101
102
103
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012
FL1-H
FL
4-H
1.07 42.8
6.6249.5
100
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102
103
104
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102
103
104
007
FL1-H
FL
4-H
1.82 0.619
0.14397.4
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103
104
008
FL1-H
FL
4-H
1.15 43.7
7.5147.6
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103
104
011
FL1-H
FL
4-H
0.633 0.586
0.24198.5
1.82 0.619
97.4 0.143
1.15 43.7
47.6 7.51
0.633 0.586
98.5 0.241
1.07 42.8
49.5 6.62
ZEBRA
p-S
824 K
AP
1
NaB- NaB+
p-S
824 K
AP
1
KAP1 is phosphorylated at Serine 824 upon virallytic activation in EBV+ BL cells
C
p-S824 KAP1 ZEBRA DAPI
p-S824 KAP1 EA-D DAPI
Xiaofan Li, et al., PLOS Pathogens, 2017
36kD
50kD
98kD
98kD
ZEBRA
β-actin
KAP1
FLAG
0h 48h
1.00 0.76 0.72 1.25
NaB
EVKAP1
wt
KAP1
S824A
KAP1
S824D
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Figure 4
EVKAP1
wt
KAP1
S824A
KAP1
S824D
Phosphorylated KAP1 at S824 is impaired in its ability to restrain EBV lytic activation
C
H
Alanine
Phospho-dead phosphomimetic phosphorylated
Xiaofan Li, et al., PLOS Pathogens, 2017;
FLAG-KAP1-wt
Empty vector
FLAG-KAP1-S824A
FLAG-KAP1-S824D
How is KAP1 phosphorylated at S824?
PS824
KAP1KAP1ATM mTOR
HCMV lytic activationDNA damage response
David White et.al, Cancer Res, 2006;
Yael Ziv et.al, Nature Cell Biology, 2006Benjamin Rauwel et al., Elife, 2015
ATM is responsible for phosphorylation of KAP1 at S824 during EBV lytic activation
A
C
p-S824 KAP1
KAP1
β-actin
ATM
98kD
NaB - + - +
scrambled si-ATM
1.0 0.63
98kD
250kD
50kD
p-S824 KAP1
KAP1
β-actin
ZEBRA
KU-55933 (μM) 0 0.1 0.5 1.0 0 0.1 0.5 1.0
-NaB +NaB
98kD
98kD
36kD
50kD
p-S824 KAP1
KAP1
β-actin
ZEBRA
Torin1 (μM) 0 0.1 0.2 0.5 0 0.1 0.2 0.5
-NaB +NaB
98kD
98kD
36kD
50kD
B
Torin1
KU
p-S824 KAP1 ZEBRA DAPI
-
Xiaofan Li, et al., PLOS Pathogens, 2017
KU-55933: ATM inhibitor
Torin1: mTOR inhibitor
Input R-IgG ATM KAP1 Input R-IgG ATM KAP1
IP IP
ATM
KAP1
IgG hc
250kD
98kD
50kD
-NaB +NaB
DAPI
BA
PLA (ATM/KAP1) ZEBRA DAPI
DAPIPLA (ATM/R-IgG) ZEBRA
PLA (G-IgG/KAP1) ZEBRA
ATM binds to KAP1 during EBV lytic activation
Xiaofan Li, et al., PLOS Pathogens, 2017
KAP1KAP1 S824
p
ZEBRA
ATMViral lytic product
(IE, E, L)
EBV genome EBV genome
Viral lytic product
(IE, E, L)
i. ii.
Working model
Chloroquine
F
ATM activator chloroquine induces phosphorylation of KAP1 at S824 and EBV lytic activation.
C
CQ 0h 4h 8h 16h 24h 48h 72h
ZEBRA
β-actin
p-S824 KAP1
KAP1
36kD
98kD
98kD
50kD
B
ZEBRA
β-actin
KAP1
CQ - + - +
KAP1
wt
KAP1
S824A
1.0 0.48
98kD
50kD
36kD
A
-
CQ
ZEBRA DAPIp-S824 KAP1
CQ
+KU
BZ
LF
1m
RN
A
(rela
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mo
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0
4
8
12
0
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20
30
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RF
1m
RN
A
(rela
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mo
un
t)
0
2.5
5.0
7.5
BF
RF
3m
RN
A
(rela
tiv
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mo
un
t)
p<0.01 p<0.01 p<0.01
D E
2700
2600
2500
400
300
100
0
200
Vir
al lo
ad
(10
6/m
l)
NDG
ZEBRA
β-actin
CQ 0h 8h 24h 72h4h 16h 48h
36kD
50kD
0h 8h 24h 72h4h 16h 48h
Jijoye
Raji
ZEBRA
β-actin
CQ
36kD
50kD
Xiaofan Li, et al., PLOS Pathogens, 2017;
10μM CQ
Mock
NaB
200μM CQ
ZEBRA DAPIgH2AX
-
NaB
NaB
+KU
Etoposide
A
B
NaB EtoposideCQ
gH2AX
DAPI
C
-
gH2AX ZEBRA DAPI
CQ
+KU
CQ
Chloroquine and NaB activate ATM in the absence of observable DNA damage
Xiaofan Li, et al., PLOS Pathogens, 2017
Conclusion
The cancer-causing virus EBV exploits a cellular mechanism (involving ATM and KAP1)
that repairs breaks in heterochromatin to promote its own replication